The long-term objective of this work is to elucidate the mechanism through which trypanosomatid mitochondrial mRNAs are edited. Over half of the genetic code in some mRNAs is created after transcription through the insertion and deletion of uridines (Us). Part of the information specifying the location and nature of the editing is contained within guide RNAs (gRNAs), but the catalytic molecules have not yet been identified. An existing in vitro system that supports gRNA-dependent U-insertions will be further optimized and used to characterize the reaction. The initial focus of the proposal will be to identify the sequence and structure of the gRNA and mRNA that support the interaction(s) with the molecules catalyzing the U-insertions. This will be accomplished through several different approaches that utilize both selection-amplification and chemical modification. RNA affinity ligands will also be generated that will be used together with conventional chromatography to purify the interacting editing components. These studies are designed to increase understanding of this ancient genetic process and to determine how it is related to other RNA processing reactions. Identification of the editing components would not only be an important step in determining the mechanism of the reaction, but it would also identify future targets for the design of drugs against some of the trypanosomatid parasites. More than 30 million people in the tropics and subtropics suffer from the diseases caused by these parasites.